Bangladesh Pharmaceutical Journal 16(2): 205-209, 2013

Preliminary Biological Investigations of fimbriatum and Calophyllum inophyllum

Md. Al Amin Sikder1, Razib Saha2, Md. Rokibuzzaman2, Tasnuva Sharmin2, Ridwan Bin Rashid3, Mohammad Zashim Uddin4 and Mohammad A. Rashid1

1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh 2Department of Pharmacy, State University of Bangladesh, Dhaka-1205, Bangladesh 3Department of Microbiology, University of Dhaka, Dhaka-1000, Bangladesh 4Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract The crude methanol extracts of the leaf of Lophopetalum fimbriatum (non Wight) F. Vill. and Calophyllum inophyllum L. as well as their pet-ether, carbon tetrachloride, chloroform and aqueous soluble fractions were evaluated for antioxidant, cytotoxic, thrombolytic, membrane stabilizing and antimicrobial activities. The antioxidant potential was evaluated by DPPH and Folin-Ciocalteau reagent using butylated hydroxytolune (BHT) and ascorbic acid as standards, respectively. Among the extractives of L. fimbriatum and C. inophyllum, the chloroform soluble fraction and methanolic crude extract demonstrated the highest free radical scavenging activity

(IC50 = 175.57 ± 0.02 µg/ml and IC50 = 1.0 ± 0.22 µg/ml) which could be correlated with their total phenol contents 82.15 ± 0.89 and 32.19 ± 0.81 mg of GAE /g of extractives, respectively. In the brine shrimp lethality bioassay, the

carbon tetrachloride soluble fractions of L. fimbriatum (LC50 = 0.515 ± 0.03 µg/ml) and C. inophyllum (LC50 = 0.77 ± 0.18 µg/ml) revealed general toxicity. During assay for thrombolytic activity, the carbon tetrachloride soluble materials of L. fimbriatum and the chloroform soluble fraction of leaf of C. inophyllum revealed clot lysis by 8.89 ± 1.410% and 27.84 ± 0.94%, while the standard streptokinase and water, used as positive and negative controls, demonstrated 66.77% and 3.79% clot lysis, respectively. In hypotonic solution and heat induced conditions, the crude methanol extracts of L. fimbriatum and C. inophyllum inhibited haemolysis of human erythrocyte by 68.14 ± 2.05% & 40.00 ± 1.6% and 57.67 ± 0.26% and 28.12 ± 0.38%, respectively. Here, acetyl salicylic acid (0.1 mg/ml) was used as reference showing 72.79% and 42.12% haemolysis of RBCs in hypotonic solution and heat induced conditions, respectively. The antimicrobial activity was assessed by the disc diffusion method and the chloroform soluble fraction of L. fimbriatum demonstrated 16.0 mm zone of inhibition against Sarcina lutea. Different extractives of C. inophyllum inhibited microbial growth with zone of inhibition ranging from 10.0 mm to 22.0 mm. Among the different extractives of C. inophyllum, the pet-ether and carbon tetrarchloride soluble fractions demonstrated 22.0 mm zone of inhibition against Vibrio parahaemolyticus and Pseudomonas aeruginosa, respectively. Key words: Lophopetalum fimbriatum (non Wight) F. Vill., Calophyllum inophyllum L., antioxidant, DPPH, cytotoxicity, thrombolytic, membrane stabilizing, zone of inhibition.

Introduction evergreen tree belonging to Calophyllaceae family. It is Lophopetalum fimbriatum (non Wight) F. Vil. native from East Africa, Southern coast of India, (Synonyms: Lophopetalum javanicum (Zoll.) Turcz., and Australia. In Bangladesh, the is distributed in Lophopetalum intermedium Ridl., Bengali name: Raktan) coastal forests of the country, especially Noakhali, Bhola, is a small tree belonging to the family . The Sandwip and Patuakhali. Bark is astringent. Pounded bark plant is distributed in Thailand, Peninsular Malaysia, is used topically in orchitis and internal haemorrhages Sumatra, Java, Borneo, , Celebes, Moluccas while its juice is used as purgative. Decoction of the bark and New Guinea. The bark is used as a constituent of dart is employed as a lotion for indolent ulcers. The gum resin poison (www.asianplant.net). is considered emetic, purgative, vulnerary, resolvent and Calophyllum inophyllum L. (Synonyms: Calophyllum anodyne and is applied to ulcers and wounds. Leaves are blumei Wight., Balsamaria inophyllum Lour., Bengali applied to sore eyes. Seed oil is applied externally in name: Punnag) commonly called Alexandrian laurel, is an rheumatism and it is a reputed antipsoric. Seed oil is also Correspondence to: Mohammad A. Rashid; Tel.: 880-2-9661900/8137; Fax: 880-2-8615583; E-mail: [email protected] 206 Sikder et al. / Bangladesh Pharmaceutical Journal 16(2): 205-209, 2013 used in gonorrhoea, gleet and scabies (Medicinal synthetic antioxidants, butylated hydroxytoluene (BHT) Database of Bangladesh). and ascorbic acid as positive controls.

As part of our ongoing investigations on medicinal Table 1. Kupchan partionates of L. fimbriatum and C. plants of Bangladesh (Kaisar et al., 2011 and Sharmin inophyllum. et al., 2012), the crude methanol extracts of leaves of L. Crude extract/ L. fimbriatum C. inophyllum fimbriatum and C. inophyllum growing in Bangladesh as Fractions (g) (g) well as their organic and aqueous soluble fractions were Me 5.0 5.0 PESF 0.5 0.5 studied for the antioxidant potential in terms of total CTCSF 1.5 1.0 phenolic content and free radical scavenging property; CSF 0.5 1.5 cytotoxic, thrombolytic, membrane stabilizing and AQSF 1.5 1.5 antimicrobial activities for the first time and we, here in, ME= Methanolic crude extract; PESF= Pet-ether soluble fraction; CTCSF= Carbon tetrachloride soluble fraction; CSF= Chloroform report the results of our preliminary investigations. soluble fraction; AQSF= Aqueous soluble fraction.

Brine shrimp lethality bioassay: This technique was Materials and Methods applied for the determination of general toxic properties of Plant materials: The leaves of L. fimbriatum and C. the DMSO solutions of plant extractives against Artemia inophyllum were collected from Mirpur Botanical garden, salina in a one day in vivo assay (Meyer et al., 1982). Dhaka in November 2011. Voucher specimens DACB- Vincristine sulphate was used as positive control. 24336 and DACB-37785 for L. fimbriatum and C. Thrombolytic activity: The thrombolytic activity was inophyllum, respectively have been maintained in evaluated by the method developed by Prasad et al. (2006) Bangladesh National Herbarium, Dhaka Bangladesh for by using streptokinase as positive control. future references. Membrane stabilizing activity: The membrane The collected plant materials were cleaned, sun dried stabilizing activity of the extractives was assessed by their and pulverized. The powdered materials (500g each) of ability to inhibit hypotonic solution and heat induced both the plants were separately soaked in 2.0 liters of haemolysis of human erythrocytes following the method methanol at room temperature for 7 days. The extracts developed by Omale et al. (2008). were filtered through fresh cotton bed and finally with Antimicrobial screening: Antimicrobial activity was Whatman filter paper number 1 and concentrated with a determined by disc diffusion method (Bauer et al., 1966). rotary evaporator at reduced temperature (40-45ºC) and pressure. An aliquot (5g) of each of the concentrated Statistical analysis: For all bioassays, three replicates methanol extracts was fractionated by the modified of each sample were used for statistical analysis and the Kupchan partitioning protocol (VanWagenen et al., 1993) values are reported as mean ± SD. and the resultant partitionates were evaporated to dryness Results and Discussion with rotary evaporator to yield pet-ether (PESF), carbon tetrachloride (CTCSF), chloroform (CSF) and aqueous The aim of the study was to evaluate the crude (AQSF) soluble materials (Table 1). The residues were methanol extracts of L. fimbriatum and C. inophyllum as then stored in a refrigerator until further use. well as their pet-ether, carbon tetrachloride, chloroform and aqueous soluble fractions for antioxidant potential in Total phenolic content: The total phenolic content of terms of total phenolic content and free radical scavenging the extractives was determined with Folin Ciocalteau property as well as cytotoxic, thrombolytic, membrane reagent by using the method developed by Harbertson and stabilizing and antimicrobial potential. Spayd (2006). In DPPH free radical scavenging assay, all the test DPPH free radical scavenging assay: Following the samples of L. fimbriatum demonstrated mild free radical method developed by Brand-Williams et al. (1995), the scavenging potential with IC values ranging from 175.57 antioxidant activity of the test samples was assessed by 50 µg/ml to 619.0 µg/ml. The highest free radical scavenging determining the scavenging activities of the stable 1,1- activity was demonstrated by the chloroform soluble diphenyl-2-picrylhydrazyl (DPPH) free radical by using fraction (IC50= 175.57 ± 0.02 µg/ml) which can be

Sikder et al. / Bangladesh Pharmaceutical Journal 16(2): 205-209, 2013 207 correlated to its phenolic content (82.15 ± 0.89 mg of induced haemolysis of RBCs. The extractives of C. GAE / g of extractives). Highly significant free radical inophyllum significantly protected the haemolysis of RBC scavenging potentials were demonstrated by all the test induced by hypotonic solution and heat. The methanolic samples of C. inophyllum with IC50 values ranging from crude extract and the carbon tetrachloride soluble fraction 1.0 µg/ml to 13.0 µg/ml. The highest free radical inhibited 57.67 ± 0.26% & 28.12 ± 0.38% and 48.29 ± scavenging activity was revealed by the crude methanol 0.01% and 14.43 ± 0.71% hypotonic solution and heat extract (IC50= 1.0 ± 0.22 µg/ml) which could be correlated induced haemolysis of RBC as compared to 72.79% and to its phenolic content 32.19 ± 0.81 mg of GAE/g of 42.12% by acetyl salicylic acid, respectively (Table 4). extractives (Table 2). L. fimbriatum and C. inophyllum test samples were All the test samples of L. fimbriatum and C. screened against five gram positive and eight gram inophyllum displayed significant cytotoxic potential negative bacteria and one fungus their microbial growth against A. salina. The carbon tetrachloride soluble inhibitory potentials. The crude methanol extract and the fractions of L. fimbriatum and C. inophyllum exhibited the chloroform soluble fraction of L. fimbriatum demonstrated highest cytotoxic activity with LC50 values 0.515 ± 0.03 zone of inhibition ranging from 8.0 to 16.0 mm. The µg/ml and 0.77 ± 0.18 µg/ml, respectively as compared to highest zone of inhibition (16 mm) was shown by the 0.451 µg/ml for Vincristine sulphate (Table 2). chloroform soluble fraction against Sarcina lutea. The The extractives of L. fimbriatum demonstrated weak crude methanol extract and its chloroform soluble fraction thrombolytic activity. The carbon tetrachloride soluble exhibited 15 mm zone of inhibition against S. lutea and fraction showed 8.89 ± 1.410% clot lysis as compared to Shigella boydii, respectively (Table 5). On the other hand, 66.77% clot lysis exhibited by the standard streptokinase. the test samples of C. inophyllum displayed zone of The extractives of C. inophyllum demonstrated mild to inhibition ranging from 10.0 to 22.0 mm. The highest zone moderate thrombolytic activity. The chloroform soluble of inhibition (22.0 mm) was observed by the pet-ether and fraction and the crude methanol extract showed 27.84 ± carbon tetrarchloride soluble fractions against Vibrio 0.94 % and 27.38 ± 1.03 % clot lysis, respectively parahaemolyticus and Pseudomonas aeruginosa, (Table 3). respectively. The chloroform soluble fraction also showed At concentration 1.0 mg/ml, L. fimbriatum extractives strong zone of inhibition (21.0 mm) against Sh. boydii and possess significant membrane stabilizing activity as V. parahaemolyticus (Table 6). Standard antibiotic, compared to the standard acetyl salicylic acid (0.10 Ciprofloxacin was involved as the reference standard in mg/ml). The crude methanol extract inhibited 68.14 ± 2.05 this assay. % and 40.00 ± 1.60 % hypotonic solution and heat

Table 2. The total phenolic content, free radical scavenging and cytotoxic activities of L. fimbriatum and C. inophyllum.

Plants Samples/ Standards Total phenolic content DPPH Free radical Cytotoxic activity (mg of GAE/g of scavenging activity (LC50 µg/ml) extract) (IC50 µg/ml) L. fimbriatum ME 80.10 ± 0.74 249.55 ± 0.57 0.731 ± 0.22 PESF 11.89 ± 0.95 619.0 ± 0.77 0.856 ± 0.68 CTCSF 15.88 ± 0.83 562.69 ± 0.38 0.515 ± 0.03 CSF 82.15 ± 0.89 175.57 ± 0.02 6.79 ± 1.01 AQSF 28.10 ± 0.73 423.10 ± 0.18 73.18 ± 0.37 C. inophyllum ME 32.19 ± 0.81 1.0 ± 0.22 1.93 ± 0.77 PESF 11.45 ± 1.24 3.95 ± 0.34 1.12 ± 0.41 CTCSF 18.24 ± 0.47 1.71 ± 0.59 0.77 ± 0.18 CSF 12.31 ± 0.39 3.0 ± 0.11 7.97 ± 0.69 AQSF 9.60 ± 0.24 13.0 ± 1.21 1.83 ± 0.51 Standards VS - - 0.451 BHT - 27.5 ± 0.54 - Ascorbic acid - 5.8 ± 0.21 -

BHT= Butylated hydroxytolune; VS= Vincristine sulfate; ME= Methanolic crude extract; PESF= Pet-ether soluble fraction; CTCSF= Carbon tetrachloride soluble fraction; CSF= Chloroform soluble fraction; AQSF= Aqueous soluble fraction

208 Sikder et al. / Bangladesh Pharmaceutical Journal 16(2): 205-209, 2013

Table 3. Thrombolytic activity of L. fimbriatum and C. inophyllum extractives.

% of lysis of RBC Sample L. fimbriatum C. inophyllum ME 4.64 ± 1.200 27.38 ± 1.03 PESF 1.945 ± 0.818 23.96 ± 0.23 CTCSF 8.89 ± 1.410 24.23 ± 0.48 CSF 2.963 ± 0.352 27.84 ± 0.94 AQSF 1.00 ± 0.906 14.08 ± 0.55 Water 3.79 ± 0.55 SK 66.77 ± 1.08

ME = Methanolic crude extract; PESF = Pet-ether soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform soluble fraction; AQSF = Aqueous soluble fraction; SK =Streptokinase

Table 4. Effect of different extractives of leaf of L. fimbriatum and C. inophyllum on hypotonic solution- and heat-induced haemolysis of erythrocyte membrane.

% Inhibition of haemolysis Sample L. fimbriatum C. inophyllum Hypnotic solution Heat Hypnotic solution Heat induced induced induced induced ME 68.14 ± 2.05 40.00 ± 1.60 57.67 ± 0.26 28.12 ± 0.38 PESF 14.08 ± 2.06 19.00 ± 0.71 42.14 ± 0.33 13.55 ± 0.82 CTCSF 18.36 ± 1.63 30.40 ± 1.43 48.29 ± 0.01 14.43 ± 0.71 CSF 66.33 ± 0.88 32.46 ± 1.63 38.6 ± 0.64 4.23 ± 0.37 AQSF 1.43 ± 0.80 16.05 ± 0.76 30.61 ± 0.49 16.05 ± 0.76 ASA 72.79 ± 0.47 42.12 ± 0.23 72.79 ± 0.47 42.12 ± 0.23

ME = Methanolic crude extract; PESF = Pet-ether soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform soluble fraction; AQSF = Aqueous soluble fraction; ASA= Acetyl salicylic acid

Table 5. Antimicrobial activity of L. fimbriatum.

Diameter of zone of inhibition (mm) Test microorganisms Ciprofloxacin ME CSF (30µg/disc) Bacillus cereus 8.0 ± 1.65 9.0 ± 1.53 45.0 ± 2.01 B. megaterium 14.0 ± 1.53 15.0 ± 1.53 42.0 ± 1.17 B. subtilis 10.0 ± 2.5 12.0 ± 3.06 42.0 ± 0.73 Staphylococcus aureus 11.0 ± 12.55 13.0 ± 2.08 - Sarcina lutea 15.0 ± 2.35 16.0 ± 1.52 42.0 ± 0.56 Escherichia coli 13.0 ± 2.55 14.0 ± 1.52 42.0 ± 0.43 Pseudomonas aeruginosa 11.0 ± 1.65 12.0 ± 2.52 42.0 ± 1.11 Salmonella Typhi - - 45.0 ± 0.73 S. Paratyphi 11.0 ± 3.55 13.0 ± 2.52 47.0 ± 2.33 Shigella boydii 14.0 ± 2.55 15.0 ± 1.53 34.0 ± 0.58 Sh. dysenteriae 11.0 ± 3.25 12.0 ± 2.08 42.0 ± 0.22 Vibrio mimicus 10.0 ± 1.75 11.0 ± 2.51 40.0 ± 0.45 V. parahaemolyticus 9.0 ± 2.75 11.0 ± 3.05 35.0 ± 0.44 Saccharomyces cerevisiae 9.0 ± 2.25 10.0 ± 1.52 38.0 ± 0.49

ME = Methanol extract; CSF = Chloroform soluble fraction

Sikder et al. / Bangladesh Pharmaceutical Journal 16(2): 205-209, 2013 209

Table 6. Antimicrobial activity of C. inophyllum.

Diameter of zone of inhibition (mm) Test Ciprofloxacin Microorganisms ME PESF CTCSF CSF (30µg/disc) Bacillus cereus 14.0 ± 0.13 16.0 ± 0.26 16.0 ± 0.08 14.0 ± 0.36 45.0 ± 2.01 B. megaterium 14.0 ± 0.34 16.0 ± 0.71 14.0 ± 0.38 16.0 ± 0.61 42.0 ± 1.17 B. subtilis 14.0 ± 0.44 18.0 ± 0.43 18.0 ± 0.19 16.0 ± 0.43 42.0 ± 0.73 Sarcina lutea 14.0 ± 0.81 14.0 ± 0.54 14.0 ± 0.47 13.0 ± 0.38 42.0 ± 0.56 Escherichia coli 14.0 ± 0.06 18.0 ± 0.44 16.0 ± 0.29 14.0 ± 0.84 42.0 ± 0.43 Pseudomonas aeruginosa 16.0 ± 0.12 14.0 ± 0.61 22.0 ± 0.19 14.0 ± 0.11 42.0 ± 1.11 Salmonella Typhi 14.0 ± 0.28 18.0 ± 0.17 20.0 ± 0.77 14.0 ± 0.24 45.0 ± 0.73 S. Paratyphi 12.0 ± 0.60 - - - 47.0 ± 2.33 Shigella boydii 12.0 ± 0.33 14.0 ± 0.69 16.0 ± 0.90 21.0 ± 0.03 34.0 ± 0.58 Sh. dysenteriae 14.0 ± 0.48 14.0 ± 0.78 16.0 ± 1.03 16.0 ± 0.44 42.0 ± 0.22 Vibrio mimicus 10.0 ± 0.02 16.0 ± 0.13 18.0 ± 0.53 16.0 ± 0.38 40.0 ± 0.45 V. parahaemolyticus 12.0 ± 0.57 22.0 ± 0.33 16.0 ± 0.76 21.0 ± 0.55 35.0 ± 0.44 Sacharomyces cerevasiae 14.0 ± 0.38 16.0 ± 0.52 14.0 ± 0.61 16.0 ± 0.74 38.0 ± 0.49

ME = Methanolic crude extract; PESF = Pet-ether soluble fraction; CTCSF = Carbon tetrachloride soluble fraction; CSF = Chloroform soluble fraction

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